CD8+ cytotoxic T lymphocytes (CTLs) are critical for the elimination of virally-infected cells, and defects in CTL responses can lead to primary immunodeficiencies and secondary lymphoproliferative syndromes. One such defect is caused by mutations in the gene encoding Inducible T cell Kinase (ITK), a kinase that serves as an amplifier of T cell receptor (TCR) signaling. Patients with mutations in ITK develop lymphoproliferative disease associated with susceptibility to viral infections. We found CTLs from ITK-deficient mice exhibit impaired killing of multiple different targets, indicating that ITK-deficiency leads to global defects in cytolysis. Treating WT CTLs with an ITK-specific inhibitor during cytolysis assays could reproduce impaired killing, suggesting that these defects were not necessarily due to altered T cell development or CTL differentiation. To further evaluate this killing defect, we examined the discrete steps involved in CTL activity, including TCR-triggered adherence to cells, immunological synapse formation, centrosome polarization, and degranulation inducing cytolysis in targets. Although early events following TCR-mediated target cell engagement, such as actin ring formation and polarization, were intact in ITK-deficient CTLs, we found defects in degranulation, suggesting ITK may play an unappreciated role in the final stages of killing. Nonetheless, prolonged culture of ITK-deficient CTLs in IL-2 could rescue defects in degranulation, similar to observations in NK cells from certain primary immunodeficiencies in which cytotoxicity is enhanced in culture after IL-2 stimulation. Together these experiments provide clues to novel roles for ITK and TCR signaling in regulating late stages of cytolysis, and further insight into the defects that may account for the susceptibility to viral infections observed in patients with mutations in ITK and TCR signaling components.
In parallel work, we also examined the role of actin in regulating degranulation in normal CTLs. While previous work showed a reduction in actin density at the synapse prior to secretion of lytic granules, we found that cortical actin recovers concomitant with the termination of secretion. Disruption of this actin network via treatment with an actin depolymerization agent resulted in a resumed degranulation, suggesting that actin acts as a reversible barrier to prevent lytic granule exocytosis. Furthermore, we provide evidence that degranulation is required to reestablish the actin barrier. Our results suggest that actin is both regulated by, and regulates, degranulation in CTLs. Experiments further revealed a correlation between the recovery of actin and phosphatidylinositol 4,5-biphosphate (PIP2) at the synapse, suggesting that the distribution of phosphatidylinositols in the membrane represent a potential mechanism through which CTLs regulate the density of cortical actin during cytolysis. Our work provides insight into actin-related mechanisms regulating secretion in CTLs, which may preserve serial killing capacity during immune responses
